1. Field of the Invention
The invention relates to soluble forms derived from the LAG-3 membrane protein which are useful as immunosuppressants, as well as antibodies capable of preventing the specific binding of the LAG-3 protein to MHC (major histocompatibility complex) Class II molecules as immunostimulants.
2. Description of the Related Art
In WO-A 91/10682, a protein designated LAG-3 has been described.
The LAG-3 protein is a protein selectively expressed by NK cells and activated T lymphocytes. Similarity of the amino acid sequence, the comparative exon/intron organization and the chromosomal localization shown that LAG-3 is related to CD4. The initial characterization of the LAG-3 gene has been described by TRIEBEL et al. (1).
The corresponding DNA codes for a type I transmembrane protein of 498 amino acids containing 4 extracellular sequences of the immunoglobulin type. LAG-3 is a member of the immunoglobulin superfamily.
The mature protein comprises 476 amino acids (SEQ ID NO:1) with a theoretical molecular weight of 52 kD. The extracellular region contains 8 cysteine residues and 4 potential N-glycosylation sites. By Western blot analysis, it was shown that LAG-3 inside PHA-blasts or activated NK cells has an apparent mass Mr of 70,000. After treatment with N-glycosidase F, a reduction in size to 60 kD was obtained, thereby demonstrating that native LAG-3 is glycosylated. Fuller details are described in WO-A 91/10682.
BAIXERAS et al., in J. Exp. Med. 176, 327-337 (2), have, in addition, described their finding that rosette formation between cells transfected with LAG-3 (expressing LAG-3 at their surface) and B lymphocytes expressing MHC Class II was specifically dependent on LAG-3/MHC Class II interaction.
Surprisingly, this ligand for MHC Class II was detected with higher levels on activated CD8.sup.+ lymphocytes (MHC Class I-restricted) than on activated CD4.sup.+ lymphocytes. In vivo, only a few disseminated LAG-3.sup.+ cells (MHC Class II-restricted) were to be found in non-hyperplastic lymphoid tissue comprising the primary lymphoid organs, that is to say thymus and bone marrow. LAG-3.sup.+ cells were to be found in hyperplastic lymphoid nodules and tonsils, as well as among peripheral blood mononuclear cells (PBMC) of patients receiving injections of high doses of IL-2.
These observations confirm that LAG-3 is an activation antigen in contrast to CD4 expressed in an subpopulation of resting lymphocytes and other cell types, in particular macrophages.
The MHC comprises Class I and Class II molecules which are membrane glycoproteins which present fragments of protein antigens to the T lymphocyte receptors (TCR). Class I molecules are responsible for the presentation to CD8.sup.+ cytotoxic cells of peptides derived in large part from endogenously synthesized proteins, while Class II molecules present to CD4.sup.+ helper lymphocytes peptides originating in the first place from foreign proteins which have entered the endocytic, that is to say exogenous, pathway. T helper lymphocytes regulate and amplify the immune response, while cytotoxic lymphocytes are needed to destroy cells irrespective of the tissues expressing "non-self" antigens, for example viral antigens. The mechanism of recognition involves intracellular signals leading to an effective activity of T lymphocytes.
It is apparent that, to initiate an immune response mediated by T (CD4.sup.+) lymphocytes, the foreign antigens must be captured and internalized in the form of peptides by specialized cells, the antigen presenting cells (APC). The resulting antigenic peptides are re-expressed at the surface of the antigen presenting cells, where they are combined with MHC Class II molecules. This MHC Class II/peptide complex is specifically recognized by the T lymphocyte receptor, resulting in an activation of the T helper lymphocytes.
Moreover, animal models created by recombination techniques have made it possible to emphasize the part played in vivo by MHC Class II molecules and their ligands.
Thus, mice deficient in MHC Class II molecules (3) and possessing almost no peripheral CD4.sup.+ T lymphocytes and having only a few immature CD4.sup.+ lymphocytes in the thymus have proved to be completely incapable of responding to T-dependent antigens.
CD4.sup.- /.sup.- mutant mice (4) have a substantially decreased T lymphocyte activity but show normal development and function of the CD8.sup.+ T lymphocytes, demonstrating that the expression of CD4 on the daughter cells and CD4.sup.+ CD8.sup.+ thymocytes is not obligatory for the development. Compared to normal mice, these CD4-deficient mice have a large amount of CD4.sup.- CD8.sup.- cells.
These doubly negative cells are restricted to MHC Class II and capable of recognizing the antigen.
When they are infected with Leishmania, these mice show a population of functional T helper lymphocytes despite the absence of CD4. These cells are restrictive to MHC Class II and produce interferon-.gamma. when they are activated by the antigen. This indicates that the lineage of the T lymphocytes and their peripheral function need not necessarily depend on the function of CD4.
It is not recognized that the proteins encoded by MHC Class II region are involved in many aspects of immune recognition, including the interaction between different lymphoid cells such as lymphocytes and antigen presenting cells. Different observations have also shown that other mechanisms which do not take place via CD4 participate in the effector function of T helper lymphocytes.
These different observations underline the pivotal role played by MHC Class II and its ligands in the immune system.
Moreover, the importance is known of chimeric molecules composed of the extracytoplasmic domain of proteins capable of binding to ligands and a constant region of human immunoglobulin (Ig) chains for obtaining soluble forms of proteins and of cell receptors which are useful, in particular, as therapeutic agents.
Thus, soluble forms of CD4 have proven their efficacy in inhibiting an HIV infection in vitro in a dose-dependent manner.
Nevertheless, clinical trials with soluble CD4 molecules, in particular of CD4-Ig, have not enabled a significant decrease in viral titres to be demonstrated. Transgenic mice expressing up to 20 .mu.g/ml of soluble CD4 in their serum were created. These mice showed no difference as regards their immune function relative to control mice. Hitherto, no direct binding to MHC Class II of molecules derived from CD4 has been reported. This strongly suggests that soluble CD4 molecules do not interact in vivo with MHC Class II molecules.